By studying the chemical reactions that occur inside particular cells, scientists can learn more about those cells. It is difficult to conduct experiments inside cells, however. One technique shown to be useful is to develop a probe to enter the cell, react with a particular substance inside the cell, and signal that the reaction has occurred.
Unfortunately, many tests for the presence of products inside of cells are destructive to the cells being tested, either killing them outright or preventing them from growing or reproducing as normal cells. They may have other disadvantages as well. For example, radioactive substances, while sufficiently sensitive to distinguish cells, ultimately destroy the viability of the cells during measurement and destroy the reproductive capability of the cells. In addition, radioactive reagents are dangerous to handle and require slow and cumbersome experimental techniques and disposal methods, and radioactive measurements can not be done on viable cells. Dyes that are chromogenic rather than fluorescent, e.g., 5-bromo-4-chloroindolyl galactoside (X-gal) and 5-bromo-4-chloro-3-indolyl .beta.-D-glucuronic acid (X-GlcU) are less sensitive and require a large turnover of substrate or multiple reactions to obtain a signal. Furthermore, the hydrolysis product of X-gal (and X-GlcU) is frequently toxic to cells.
In order to study living cells, tests are typically limited to only a portion of a cell population. If the subject cell population is a very small one, however, and some cells are removed for testing, sufficient cells may not be available for further study or use. In order to study or otherwise utilize a small population of cells, as living cells, it is essential to be able to locate and, if possible, separate those cells without destroying them.
Ideally, a probe used to identify and separate a living cell which contains a particular substance or to localize a substance in an organelle of a living cell, has the following characteristics: 1) the probe enters the cell without damaging the cell or preventing its subsequent cloning or reproduction, 2) the probe reacts exclusively with the particular substance inside the cell to form a specific detection product, 3) the detection product produces a signal sufficiently intense to distinguish the cell from other cells that do not contain the substance or that contain less of the substance, and 4) the detection product is sufficiently well retained by the cell to permit analysis and, if desired, sorting of the cell.
Fluorescent enzyme substrates generally make ideal probes. Often, a fluorescent substrate can enter the cell using the cell's own mechanisms. Once inside the cell, a fluorescent substrate usually only reacts with a specific enzyme. Typically, the reaction produces a change in fluorescence which is sufficiently distinctive to distinguish cells or organelles that have the enzyme from cells or organelles that do not or that have lower levels of the enzyme.
Use of fluorescent substrates also permits utilization of flow cytometers. Flow cytometers are designed for the rapid and specific sorting of highly fluorescent cells from cells that have low fluorescence. Flow cytometers commonly use an argon laser to excite the fluorescent product inside the cells. Thus, excitation of fluorescence at the principal wavelengths of the argon laser (488 or 514 nm) or by longer wavelength excitation sources is a preferred characteristic of a fluorescent substrate for some applications. As a result, fluorescent substrates which respond poorly at this wavelength, such as umbelliferone (7-hydroxycoumarin) conjugates, are not as suitable for such applications.
Two advantages exist for even longer wavelength probes that absorb at greater than about 500 nm: 1) the autofluorescence from cells generally decreases with increasing wavelengths and 2) longer wavelength emission can be detected in the presence of a second dye such as fluorescein that emits at a shorter wavelength, permitting measurement of two parameters that are detected simultaneously or sequentially at separate wavelengths.
Quantitative imaging of fluorescence using microscopes and image intensifiers has been used to measure substances such as intracellular calcium ions and superoxide production in living cells. Methods exist for quantitatively measuring changes in fluorescence intensity with time, such as occurs in turnover of fluorescent substrates. Quantitative differences in the fluorescence change that result from hydrolysis of a fluorescent substrate may be the result of either a higher enzyme content or a faster enzyme turnover rate.
Fluorescent substrates, and flow cytometry, can also be used to detect and separate cells which have acquired the ability to produce certain enzymes as a result of a gene fusion. Gene fusions are used to study or work with a particular gene or genetic material by inserting it into a host cell. Typically, the foreign genetic material is inserted into the host cell using a vector (transfection). Alternatively, the foreign genetic material enters the cell through pores created in the membrane, e.g. electroporation, or is microinjected into the host cell. Cells which have successfully incorporated the foreign genetic material are termed "transformed".
One way to determine whether transformation has occurred is to test for the presence of a protein product resulting from the inserted genetic material. Depending on the nature of the foreign genetic material inserted into the host cell and the desired genetic characteristics of the transformed cell, however, testing for successful transformation can be expensive and time-consuming.
Glycosidic enzymes are commonly used to differentiate cells, including transformed cells. For example, .beta.-galactosidase is a bacterial enzyme commonly found in Escherichia coli (E. coli). The enzyme is coded by the E. coli lacZ gene. The presence of .beta.-galactosidase activity in a transformed cell can be used to indicate the presence of the foreign lacZ gene. The lacZ gene, in turn, is used as a genetic marker to indicate that additional foreign genetic material, including the lacZ gene, has been incorporated into a host cell otherwise lacking in .beta.-galactosidase. The enzyme .beta.-glucuronidase, coded by the GUS gene in E. coli, is primarily used to detect transformation in plant cells and tissues, where this activity is normally lacking (Jefferson, The GUS Reporter Gene System, NATURE 342, 837 (1989)), but it is also useful in detecting transformations in mammalian cells.
Not all glycosidic enzymes are useful as marker enzymes. Some glycosidic enzymes, such as .beta.-glucosidase, are intrinsically present in many cells. Their activity, however, may be characteristic of the cell type, of an organelle of the cell, or of the metabolic state of the cell. Some common glycosidic enzymes and representative carbohydrates cleaved by such enzymes are listed in Table 1. This listing is not meant to limit or define the extent of all glycosidic enzymes.
TABLE 1 ______________________________________ SELECTED GLYCOSIDIC ENZYMES (from ENZYME NOMENCLATURE, 1984 (International Union Biochemistry, Academic Press, 1984 pages 306-26) CARBOHYDRATE- GROUP E.C. NO. ENZYME SELECTIVITY ______________________________________ 3.2.1.18 Sialidase N- or O-Acetyl Neuraminic Acid (Sialic Acid) 3.2.1.20 .alpha.-Glucosidase .alpha.-D-Glucose 3.2.1.21 .beta.-Glucosidase .beta.-D-Glucose 3.2.1.22 .alpha.-Galactosidase .alpha.-D-Galactose 3.2.1.23 .beta.-Galactosidase .beta.-D-Galactose 3.2.1.24 .alpha.-Mannosidase .alpha.-D-Mannose 3.2.1.25 .beta.-Mannosidase .beta.-D-Mannose 3.2.1.26 .beta.-Fructofuranosidase .beta.-D-Fructose 3.2.1.30 N-Acetyl-.beta.-glucosaminidase .beta.-D-N-Acetyl- Glucosamine 3.2.1.31 .beta.-Glucuronidase .beta.-D-Glucuronic Acid 3.2.1.38 .beta.-D-Fucosidase .beta.-D-Fucose 3.2.1.40 .alpha.-L-Rhamnosidase .alpha.-L-Rhamnose 3.2.1.43 .beta.-L-Rhamnosidase .beta.-L-Rhamnose 3.2.1.48 Sucrose .alpha.-glucosidase .alpha.-D-Glucose 3.2.1.49 .alpha.-N-Acetylgalactosaminidase .alpha.-D-N-Acetyl- Galactosamine 3.2.1.50 .alpha.-N-Acetylglucosaminidase .alpha.-D-N-Acetyl- Glucosamine 3.2.1.51 .alpha.-L-Fucosidase .alpha.-L-Fucose 3.2.1.52 .beta.-N-Acetylhexosaminidase .beta.-D-N-Acetyl- Glucosamine 3.2.1.53 .beta.-N-Acetylgalactosaminidase .beta.-D-N-Acetyl- Galactosamine 3.2.1.55 .alpha.-L-Arabinofuranosidase .alpha.-L-Arabinose 3.2.1.76 L-Iduronidase .alpha.-L-Iduronic Acid 3.2.1.85 6-Phospho-.beta.-galactosidase 6-Phospho-.beta.-D- Galactose 3.2.1.86 6-Phospho-.beta.-glucosidase 6-Phospho-.beta.-D-Glucose 3.2.1.88 .beta.-L-Arabinosidase .beta.-L-Arabinose 3.2.1.4 Cellulase .beta.-Cellobiose ______________________________________
When the presence of an enzyme is used to indicate gene fusion, the marker included with the foreign genetic material provides a relatively fast and inexpensive means of detecting successful transformation. Cells which have successfully incorporated the marker gene are called "marker" positive (e.g. lacZ.sup.+ or GUS.sup.+). Using the marker gene to show successful transformation, however, requires detecting the activity of a very small number of enzyme molecules, usually in the cytosol of the lacZ.sup.+ or GUS.sup.+ cell. The activity must be detected in a way which does not inhibit further use, replication or study of the living transformed cell. Activity of the marker enzyme is most often used to monitor: 1) promotor and/or repressor effectiveness; 2) the crucial sequence of the promotor gene after sequential or selective deletions on it; 3) the level of induction of the operon so as to evaluate the effectiveness of potential inducer(s); and 4) any possible gene expression regulation at the pro- and/or post-transcription or translation level. Such monitoring is done by the methods generally known in the art, such as described by Jarvis, Hagen, & Sprague, Identification of a DNA segment that is necessary and sufficient for .alpha.-specific gene control in Saccharomyces cerevisiae: implications for regulation of .alpha.-specific and a-specific genes, MOLEC. & CELL. BIOL. 8, 309 (1988).
Several substrates derived from fluorescent dyes have previously been described for measurement of glycosidic activity both in cell extracts and of the purified enzyme. Among the most common fluorescent substrates for detection of galactosidase activity are .beta.-methylumbelliferyl galactoside, resorufin galactoside, fluorescein digalactoside, and Naphthol AS-BI galactoside. Fluorescent substrates for detection of glucuronidase activity include 4-methylumbelliferyl .beta.-D-glucuronic acid, resorufin .beta.-D-glucuronic acid, 4-trifluoromethylumbelliferyl .beta.-D-glucuronic acid, Naphthol AS-BI .beta.-D-glucuronide, and fluorescein mono-.beta.-D-glucuronide.
Most glycosidase substrates have been designed to be water soluble to facilitate their use in aqueous solution. This hydrophilic character appears to retard passage of the substrate through the membrane of living cells. Legler & Liedtke, Glucosylceramidase from Calf Spleen, BIOL. CHEM., 366, 1113 (1985) describe the use of fluorescent glucosidase substrates 4-heptyl-, nonyl-, and -undecylumbelliferone in assaying glucosylceramidase purified from calf spleen. Legler & Liedtke note a preference of the enzyme for long aliphatic side chains in the aglycon. The longer alkyl chains, however, appear to interfere with fluorescence and solubility in the absence of detergents. Legler & Liedtke do not use resorufin derivatives or other derivatives that have long wavelength absorption or fluorescence emission and do not discuss the assay of glucosidase inside intact living cells, or in tissues.
Although the use of fluorescent substrates is preferable to other methods, such as radioactivity, they are not entirely problem-free. In addition to the problems of cell leakage and cell entry discussed in greater detail below, some of the disadvantages of these substrates include fluorescence at a wavelength not well suited for flow cytometry (e.g. .beta.-methylumbelliferyl galactoside, 4-trifluoromethylumbelliferyl galactoside, naphthol AS-BI galactoside), pH sensitivity or pH change necessary to exhibit maximal fluorescence (e.g. .beta.-methylumbelliferyl galactoside), and low sensitivity or limited change in fluorescence in the presence of the enzyme (e.g. naphthol AS-BI galactoside).
U.S. Pat. No. 4,812,409 to Babb et al. (1989) discloses substrates attached to a blocked phenalenone or benzphenalenone fluorescent moiety, which when cleaved from the substrate by hydrolysis at a pH of 9 or less, releases a fluorescent moiety excitable at a wavelength above about 530 nm with maximum fluorescent emission at a wavelength of at least about 580 nm. There is no indication in the patent that the substrate is non-toxic to living cells or that the fluorescent product(s) do not leak from cells after enzymatic turnover, and are thus amenable to in vivo detection of enzyme activity.
Fluorescent compounds that are not enzyme substrates have been used to detect transformed cells. David W. Galbraith, active in research involving fluorescent dyes used with plant cells, identified four dyes used to label plant cell populations prior to gene fusion in Selection of Somatic Hybrid Cells by Fluorescence-Activated Cell Sorting, in CELL CULTURE AND SOMATIC CELL GENETICS OF PLANTS 1, 433, ch. 50 (1984). The four dyes, octadecanoyl aminofluorescein (F18), octadecyl rhodamine B (R18), fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC) were nontoxic to the cells (although FITC and RITC are toxic at high levels) and did not leak from the cells during culturing (p. 434). The fluorescein dye was added to one cell population, the rhodamine dye to the other. After gene fusion, the presence of both dyes was used to detect the heterokaryons. Galbraith noted (p. 442) that the lipophilic F18 and R18 dyes were observed localized in the membranes of cells, whereas FITC and RITC were distributed through the cytoplasm. Neither set of dyes was used to identify specific enzymes associated with the cells.
An article by Nolan, et al., Fluorescence-activated cell analysis and sorting of viable mammalian cells based on .beta.-D-galactosidase activity after transduction of Escherichia coli lacZ, CELL BIOLOGY 85, 2603 (1988) describes the measurement of galactosidase activity in lacZ.sup.+ transformed cells using fluorescein di-.beta.-D-galactopyranoside (FDG). The use of FDG to measure promoter activity is described in another article, Ikenaka, et al., LABORATORY METHODS: Reliable Transient Promoter Assay Using Fluorescein-di-.beta.-D-galactopyranoside Substrate, DNA & CELL BIOL. 9, 279 (1990). FDG has excellent properties for these purposes. Despite the advantages; however, there are at least two major drawbacks that are recognized in the use of FDG and all other fluorescent substrates for the analysis and selection of transformed cells.
First, it is difficult to get the substrate through the outer cell membrane without disrupting the cell. The permeability properties of available substrates, such as 4-methyl umbelliferyl glucuronide, require detection of GUS activity in plant tissue homogenate or cell extracts. Such destructive assay conditions will certainly cause inaccuracy and set limitations when an investigator is looking for relatively rare events such as the regulation of transcription and/or translation or transformation with a chimeric gene. Nolan, et al., using FDG, reduced this problem for cells in suspension by using brief hypo-osmotic or hypotonic shock. Ikenaka, et al. used the same technique. To use hypo-osmotic shock, the cells are placed in a hypotonic solution causing the membrane to swell. Swelling of the membrane results in permeabilization of the substrate so that it enters the cell. If the cell stays too long in the dilute solution, however, it ruptures. Removal of the cells from the dilute solution must be carefully timed to maximize entry of the substrate yet minimize cell loss.
A second and more important drawback of known fluorescent substrates is the problem of cell leakage. For example, following enzymatic hydrolysis of FDG, the resulting product (fluorescein) rapidly leaks out of the cell. See, e.g., FIG. 3(c) and FIG. 4; see also, Ikenaka, et al.; Nolan, et al. Commonly half of the fluorescein leaks from the cell in about 10 minutes at about 37.degree. C. Fluorescent products derived from other substrates, including all .beta.-methylumbelliferyl and resorufin glycosides have been found to leak from cells under in vivo conditions even faster than fluorescein. Nolan et al., and Ikenaka, et al., working with FDG, were able to suppress the leakage of fluorescein by quickly cooling their cells to 4.degree. C. Cooling the cells, however, also reduces the enzyme turnover rate significantly, and is not desirable when working with whole living organisms. Leakage of the fluorescent product, even at 4.degree. C. makes enzyme activity quantitation particularly difficult. It also increases the difficulty in differentiating weakly expressing gene positive cells from the background fluorescence of negative cells.
Neither hypo-osmotic shock loading nor sudden cooling well below physiological temperatures is suitable for measuring enzyme activity in transformed living cells, tissues or organisms under physiological conditions (typically 37.degree. C.) such as during development and cell division. A copending application, LIPOPHILIC FLUORESCENT GLYCOSIDASE SUBSTRATES (Ser. No. 07/623,600, filed Dec. 7, 1990), describes lipophilic derivatives of fluorescein glycosides and related compounds that yield green fluorescent products suitable for detection of glycosidase activity in living cells. Both classes of substrates are permeant to cells under physiological conditions, are not toxic to living cells, and are nonfluorescent until specifically hydrolyzed by the glycosidase enzyme. Both of their fluorescent products are readily detected in single cells and even within specific organelles of single cells. Substrates of either class can be selected that yield fluorescent products that are very well retained in the original cell with no or minimal leakage or transfer between marker-positive and marker-negative cells, even through cell division.
The two inventions differ, however, in the use of a different fluorophore. As a result, the compounds disclosed in the co-pending application have emission that is maximal at wavelengths less than 550 nm. The resorufin derivatives of this invention, in contrast, have orange to red emission that is maximal above 550 nm. This contrast permits, for example, the simultaneous detection of two different enzymes or a combination of activity of one enzyme and a second fluorescent label in a mixture of cells or even in a single cell. It also makes possible the detection of different genetic elements under regulation of different promoters in cells or tissues.
Hofman & Sernetz, Immobilized Enzyme Kinetics Analyzed by Flow-Through Microfluorimetry, ANALYTICA CHIMICA ACTA 163, 67 (1984) describes the synthesis and enzymatic properties of resorufin .beta.-D-galactopyranoside but does not describe lipophilic alkylated derivatives of this compound or the measurement of enzyme activity inside live cells. Attempts have been made to use resorufin .beta.-D-galactoside to detect incorporation of the lacZ gene in cells, but were unsuccessful as the result of leakage of the hydrolysis product from the cell.
Leakage of 7-hydroxyresorufin resulting from the use of 7-ethoxy- and 7-pentoxyresorufin in an assay for monooxygenase activity is described by Reiners, et al., Fluorescence Assay for Per-Cell Estimation of Cytochrome P-450-Dependent Monooxygenase Activities in Keratinocyte Suspensions and Cultures, ANALYT. BIOCHEM. 188, 317 at p. 322 (1990). Leakage of metabolic products of other substrates is also described. Although Reiners, et al. note improved retention of 7-hydroxyresorufin in hepatocytes, only 29% of the intracellular product remained after 35 minutes. Furthermore, there is no indication that the activity of the enzyme they were studying (which is not a glycosidase) can be determined on a single cell basis.
U.S. Pat. No. 3,731,222 to Drexhage (1973) describes a resorufin derivative with a lower alkyl (1-6 carbons) for use as a laser dye. There is no indication in the reference that an alkylated resorufin could be attached to a carbohydrate moiety for use as a glycosidase substrate, nor that that alkyl group(s) would provide any advantage to such a substrate.
Resorufin glycosides with lower alkyl (1-5 carbons) substituents for use as glycosidase substrates are described in German Patent No. DE 3411574A1 to Klein, et al., 1985. The patent does not describe the use or the advantages of alkyl residues of more than 5 carbons to increase the time that the fluorescent hydrolysis product is retained in intact cells. In fact, the patent recites that alkyl substituents with 1-3 carbons are preferred.